Tim-3, a Novel Immune Receptor, Is Constitutively Expressed on Human Natural Killer Cells and Functions as An Activating Coreceptor

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 106-106
Author(s):  
Michelle Gleason ◽  
Todd Lenvik ◽  
Valarie McCullar ◽  
Sarah Cooley ◽  
Michael Verneris ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Cell Activation ◽  
Low Dose ◽  
Cell Function ◽  
Nk Cell ◽  
Effector Function ◽  
Advisory Committees ◽  
Immune Receptor ◽  
Ifn Γ

Abstract Abstract 106 NK cells are an attractive option for immunotherapy as they do not require pre-sensitization for anti-tumor activity and do not induce graft versus host disease (GvHD) in an allogeneic transplant setting. The potential of NK cells in controlling human hematological malignancies has been increasingly recognized in recent years, as the adoptive transfer of alloreactive NK cells in hematopoietic cell transplantation (HCT) clinical trials have demonstrated therapeutic anti-leukemia effects. NK cell function is regulated by the integration of antagonist signals received from cell surface activating and inhibitory receptors. Tim-3 is a novel immune receptor that is a member of the T cell immunoglobulin and mucin-containing domain (TIM) family of glycoproteins. While its role in T cells and antigen presenting cells has been described, little is known about its function in human NK cells. While Tim-3 is present on a variety of immune cells, resting NK cells constitutively express Tim-3 compared to other lymphocyte populations (NK: 73±3%; NKT: 6±1%; T: 1±1%; n=14) and we hypothesized that Tim-3 may be important in mediating NK cell function. The unique subset of cytokine producing CD56Bright NK cells exhibited significantly lower resting Tim-3 expression compared to CD56Dim NK cells (53±3% vs. 75±3%; p<0.001, n=14). Distinct Tim-3 expression patterns were found on resting CD56Dim NK cells and activation with low dose IL-12 (1ng/mL) and IL-18 (10ng/mL), intended to more closely mimic physiologic conditions, resulted in further differentiation of this unique expression pattern dividing NK cells into 4 distinct populations: Tim-3 was homogeneously up-regulated on all CD56Bright NK cells after activation while CD56Dim NK cells were further stratified into 3 defined populations with Tim-3hi, Tim-3lo and Tim-3neg expression. The only identified ligand of Tim-3 is galectin-9 (Gal-9), a β-galactoside binding lectin, which is expressed on a wide range of healthy and malignant cells. To investigate the potential function of Tim-3, an expression vector containing human Gal-9 was transduced into K562 and Raji cells, both without endogenous Gal-9 expression. Resting NK cytotoxicity (51Cr release) was found to be increased in the presence of Gal-9 compared to the non-Gal-9 expressing targets [E:T=0.7:1, K562 vs. K562-Gal-9: 25±3% vs. 33±3% (n=8, p<0.05); E:T=20:1, Raji vs. Raji-Gal-9: 8±1% vs. 17±2% (n=4, p<0.05)]. Analysis of CD107a degranulation showed that resting Tim-3+ CD56Bright cells were more functional against Gal-9 expressing targets than Tim-3− CD56Bright cells, suggesting that Tim-3 might also play a role in IFN-γ production. To further investigate this, resting NK cells were activated with low-dose IL-12/IL-18 overnight and IFN-γ levels were measured in response to soluble rhGal-9 (0, 2.5, 5, 10 and 20nM). Exposure to soluble rhGal-9 alone without IL-12/IL-18 did not induce IFN-γ production. For both the CD56Bright and CD56Dim IL-12/IL-18 activated NK populations, only Tim-3+ NK cells displayed a dose dependent increase in IFN-γ production upon exposure to soluble rhGal-9 compared to Tim-3− NK cells. To understand the relevance of the distinct Tim-3 populations circulating in resting blood, CD56Bright, CD56Dim/Tim-3hi, CD56Dim/Tim-3lo and CD56Dim/Tim-3neg populations were sorted, cultured overnight in IL-12/IL-18 and exposed to soluble rhGal-9. Results showed the Tim-3 expressing populations contain the predominant IFN-γ producing cells that were responsive to rhGal-9 (results for the sorted CD56Dim/Tim-3lo population shown in the figure below). This increase in IFN-γ production within the Tim-3 expressing NK cell populations was abrogated by the addition of β-lactose, a β-galactoside that binds and blocks Gal-9 activity. Lastly, Western blot and immunohistochemistry analysis of human primary acute leukemia blasts revealed high Gal-9 expression. As the presence of ligands for NK cell activating receptors on tumors provide an important prerequisite for NK cell activation and effector function, we show a novel functional role for the receptor Tim-3 in human NK cell biology in the presence of its ligand Gal-9. We, therefore, propose a model where constitutively expressed Tim-3 is up-regulated by NK cell activation and effector function is enhanced by Tim-3/Gal-9 interaction, which may potentiate the elimination of Gal-9 positive tumors by NK cells. Disclosures: Niki: GalPharma: Membership on an entity's Board of Directors or advisory committees. Hirashima:GalPharma: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Clinical Significance of KIR Genotype in Patients with Methotrexate-Associated Lymphoproliferative Disorders

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5219-5219
Author(s):  
Norina Tanaka ◽  
Aya Watanabe ◽  
Suguru Honda ◽  
Mitsuko Kobayashi ◽  
Yan-Hua Wang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Function ◽  
Nk Cell ◽  
Hla Class I ◽  
Lymphoproliferative Disorders ◽  
Advisory Committees ◽  
Kir Genes ◽  
Haplotype A

Background: Methotrexate-associated lymphoproliferative disorders (MTX-LPD) develops in patients with rheumatoid arthritis (RA) or other autoimmune disorders during low-dose MTX treatment. MTX-LPD includes wide variety disease spectrum, ranging from polymorphic proliferation to aggressive lymphoma. Although etiology of MTX-LPD has not been fully understood, approximately half of the MTX-LPD cases showed association with EB virus (EBV), suggesting that MTX treatment causes reduced immune response to EBV-positive cells, and results in MTX-LPD development. Natural killer (NK) cells play important roles in eradicating tumor and virus-infected cells. NK cell function is modulated by multiple cell surface receptors, including Killer immunoglobulin-like receptor (KIR). There are multiple KIR genes (inhibitory or activated), which are various in number and/or composition among individuals, on chromosome 19q. Previous reports demonstrated that combination of KIR genes affects NK cell function, and is associated with the risk of development of certain types of cancers, viral infections and collagen disease. There is no report about the association of KIR genotype and MTX-LPD. We consider that NK cells play a significant role in suppression of MTX-LPD development. In this study, we focused on examining genotype KIR and KIR-ligand (HLA class I). Methods: We retrospectively analyzed 35 MTX-LPD cases diagnosed between 2009 and 2019. Genomic DNA was extracted from mononuclear cells that were isolated from the bone marrow or peripheral blood samples of patients with MTX-LPD. KIR genotypes were analyzed using the KIR genotyping sequence-specific primers kit. The variations of KIR content and haplotype and their relationship with progression to malignant lymphoma (ML) and response to chemotherapy were investigated. HLA was analyzed using PCR-Luminex assay. The frequency of each HLA allele and each combination was determined by referring to the data base of an HLA laboratory. Chi-squared (χ2) tests and Wilcoxon rank sum tests were used to test associations between the variables. Results: Among the 35 patients, 25 were diagnosed with ML and 10 with polymorphic LPD. Diffuse large B cell lymphoma (DLBCL) was most common type in ML (57.7%). Table 1 showed characteristics of patients and summary of the results. All patients underwent MTX treatment for RA. The median duration of MTX administration at the time of MTX-LPD diagnosis is 11.5 years (range=0.8-27.2), and median MTX dose was 10mg/week (range=4-17.6). The duration and dose of MTX had no effect between ML and polymorphic LPD. Twenty-three patients required chemotherapy, and 12 patients had tumor regression after stopping MTX treatment. Relative patient populations requiring chemotherapy in ML or polymorphic LPD were 85% or 11%, respectively (P=0.0001). EBV-positive patients tended to regress tumors with MTX discontinuation alone (P=0.16). In KIR genotype analysis, patterns of number and combination of the KIR genes are mainly classified as haplotype "A" containing multiple inhibitory KIR genes with a KIR 2DS4 (an activated KIR [aKIR]) and haplotype "B" (other than haplotype "A"). Patients were classified in haplotype A (13 cases, 37%) and haplotype B (22 cases, 63%), respectively. ML patients showed higher ratio in haplotype A (ML 46.2% vs LPD 11.1% P=0.045). There was no difference in number of aKIR or iKIR between ML and polymorphic LPD patients. In HLA Class I analyses, there was significant difference in frequencies of HLA-C haplotype between lymphoma and polymorphic LPD patients (P=0.026). Furthermore, HLA- C1 / C1 patients were more relapsed or refractory to chemotherapy than C1 / C2 patients (P = 0.17). Conclusion: This is the first report showing clinical significance of KIR genotypes in MTX-LPD. Patients with haplotype A, a suppressive haplotype, seems to be at high risk for developing lymphomas that require chemotherapy during MTX treatment. HLA- C1/C1 patients are more likely to develop lymphomas that respond poorly to treatment, suggesting that the activity of NK cells may be lower because ligands can match with KIRs that are more restricted than C1/C2. Considering the potential NK functions with KIR genotype would improve the understanding of the prognosis and lead to prevention for MTX-LPD. Disclosures Hagiwara: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Harigai:Bristol Myers Squibb Co: Other: personal fees, Research Funding; Eisai Co: Other: personal fees, Research Funding; Ayumi Pharmaceutical Co: Other: personal fees, Research Funding; AbbVie Japan GK,: Other: personal fees, Research Funding; Eli Lilly Japan K.K: Other: personal fees; Kissei Pharmaceutical Co.: Other: personal fees; Teijin Pharma Ltd: Other: personal fees, Research Funding; Mitsubishi Tanabe Pharma Co: Research Funding; Nippon Kayaku Co.: Research Funding; Pfizer Japan Inc.: Other: personal fees; Chugai Pharmaceutical Co., Ltd.: Other: personal fees; Japan College of Rheumatology: Other: personal fees; Boehringer Ingelheim Japan, Inc: Other: personal fees; GlaxoSmithKline K.K: Other: personal fees; Oxford Immuotec,: Other: personal fees. Tanaka:Bristol-Myers Squibb: Research Funding.

Engineering of Trispecific Molecule That Induces Natural Killer Cell Expansion While Mediating CD133-Specific Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3518-3518
Author(s):  
Jörg Uwe Schmohl ◽  
Martin Felices ◽  
Jeffrey S. Miller ◽  
Dan Vallera
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cell Expansion ◽  
Nk Cell ◽  
Hematologic Malignancies ◽  
Cytokine Release ◽  
Advisory Committees ◽  
Anti Cancer ◽  
Ifn Γ

Abstract Background: Selective cancer stem cell (CSC) elimination in solid as well as in and hematologic malignancies is a critical goal in immunotherapy since CSC cause drug refractory relapse. Furthermore this small cell group is known for tumor initiation and self-renewal capabilities and was already described to be a valuable target in breast, head and neck as well as in ovarian cancer. CSCs are also important for the development of hematologic malignancies and might be an excellent cell to target, currently not addressed through current therapies, in order to prevent relapse. The design of state-of-art anti-cancer immune engagers should address three important issues including ADCC, CSC targeting, and rapid effector cell expansion that sustains a potent anti-cancer response. In order to improve the current conventional bispecific immune-engager platform, we synthesized a 16133 BiKE consisting of scFvs binding FcγRIII (CD16) on Natural Killer (NK) cells and CD133 on CSC and then introduced a modified IL-15 crosslinker capable of stimulating NK effector cells. Methods: DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE) (Figure 1A). The construct was tested for specificity using flow cytometry, cytotoxicity using chromium release assays, and lytic degranulation. IL-15-mediated expansion was measured using flow based proliferation assays. Also, Interferon (IFN)-γ release was measured by flow cytometry since it is important in the anti-cancer response. Results: The new TriKE showed specific and efficient induction of NK cell related cytotoxicity as seen in 51chromium release assays with Caco-2 cells, which express high levels of CD133. Activity was superior compared to 16133 BiKE as seen in effector:target ratios of 20:1 (21.9 ± 0.8% vs. 17.9 ± 2.2%), 10:1 (9.4 ± 0.3% vs. 7.9 ± 2.4%) and 5:1 (4.3 ± 0.2% vs. 5.4 ± 1.5%) (Figure 1B). Proliferation and NK expansion with the 1615133 TriKE was far greater than that achieved with the BiKE devoid of IL-15 as tested with purified NK cells exposed to both drugs for 7 days and stained with a reactive dye (Proliferation index 1.7 ± 0.3 vs. 1.2 ± 0.01, p=<0.0001, n=5). Drug binding and induction of cytotoxic degranulation was CD133+ specific as proved with Caco-2 and Raji cells as positive and negative controls (respectively). In Caco-2 cell targets the BiKE as well as the TriKE showed significant superior activity compared to, NCI IL-15, anti-CD16 scFv and anti-CD133 scFv controls (CD107a expression 37.5 ± 0.2% and 36.9 ± 0.2% vs. 19.6 ± 0.1, 18.3 ± 0.7, 12.6 ± 0.4, p<0.001, n=3), (Figure 1C). NK cell related cytokine release measured via IFN-γ detection was higher in the TriKE compared the BiKE (38.3 ± 0.2 % vs. 13.1 ± 0.3 %, p<0.001, n=3) and higher than all other controls. NK cell related cytokine release studies showed that although IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18 (38.3 ± 0.2 % vs. 60 ± 0.2 %, p<0.001, n=3) indicating that the release induced with the TriKE was not at supraphysiologic levels. Conclusion: 1615133 TriKE showed specific and improved anti-cancer activity over BiKE and provides a self-sustaining mechanism via induction of IL-15 signaling on NK cells. Inclusion of IL-15 might be a promising platform technology since CD133 can be substituted by other promising tumor associated antigens to create a highly specific and efficient drug. By improving NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse inducing CSC with an encouraging safety profile. Disclosures Miller: Oxis Biotech Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Conferral of Enhanced Natural Killer Cell Function by KIR3DS1 in Early Human Immunodeficiency Virus Type 1 Infection

2008 ◽  
Vol 82 (10) ◽  
pp. 4785-4792 ◽  
Author(s):  
Brian R. Long ◽  
Lishomwa C. Ndhlovu ◽  
Jorge R. Oksenberg ◽  
Lewis L. Lanier ◽  
Frederick M. Hecht ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Nk Cell ◽  
Killer Cell ◽  
Cd107a Expression ◽  
Ifn Γ ◽  
Hiv 1

ABSTRACT A flurry of recent reports on the role of activating and inhibitory forms of the killer cell immunoglobulin-like receptors (KIR) in natural killer (NK) cell activity against human immunodeficiency virus type 1 (HIV-1) have yielded widely divergent results. The role of the activating NK receptor encoded by the KIR3DS1 allele and its putative ligands, members of the HLA class I Bw4Ile80 cluster, in early HIV-1 disease is controversial. We selected 60 treatment-naïve adults for study from the OPTIONS cohort of individuals with early HIV-1 infection in San Francisco. We performed NK cell functional assays measuring gamma interferon (IFN-γ) and CD107a expression by NK cells in the unstimulated state and after stimulation by the major histocompatibility complex class I-deficient 721.221 B-lymphoblastoid cell line. In addition, we measured CD38 expression (a T-cell activation marker) on T and NK cells. Persons who have at least one copy of the KIR3DS1 gene had higher IFN-γ and CD107a expression in the unstimulated state compared to those who do not possess this gene. After stimulation, both groups experienced a large induction of IFN-γ and CD107a, with KIR3DS1 carriers achieving a greater amount of IFN-γ expression. Differences in effector activity correlating with KIR3DS1 were not attributable to joint carriage of HLA Bw4Ile80 and KIR3DS1. We detected a partial but not complete dependence of KIR3DS1 on the members of B*58 supertype (B*57 and B*58) leading to higher NK cell function. Possessing KIR3DS1 was associated with lower expression of CD38 on both CD8+ T and NK cells and with a loss or weakening of the known strong associations between CD8+ T-cell expression of CD38 mean fluorescence intensity and the HIV-1 viral load. We observed that possessing KIR3DS1 was associated with higher NK cell effector functions in early HIV-1 disease, despite the absence of HLA Bw4Ile80, a putative ligand of KIR3DS1. Carriage of KIR3DS1 was associated with diminished CD8+ T-cell activation, as determined by expression of CD38, and a disruption of the traditional relationship between viral load and activation in HIV-1 disease, which may lead to better clinical outcomes for these individuals.

scholarly journals Low-Dose Interleukin-2 Therapy Enhances Cytotoxicity of CD56bright NK Cells in Patients with Chronic Gvhd

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 606-606
Author(s):  
Tomohiro Kubo ◽  
Rizwan Romee ◽  
John Koreth ◽  
Roger Belizaire ◽  
Yusuke Kamihara ◽  
...  
Keyword(s):  
Nk Cells ◽  
Single Cell ◽  
Board Of Directors ◽  
Low Dose ◽  
Nk Cell ◽  
Cell Mass ◽  
Cytolytic Activity ◽  
Mass Cytometry ◽  
Advisory Committees ◽  
Hla Dr

Abstract Introduction: Natural killer (NK) cells play an important role in defense against infections and cancer. Two major subsets of mature NK cells have been described. CD56bright CD16- NK cells, which normally represent only 5-10% of circulating NK cells, are thought to exhibit less cytolytic activity and greater immune regulatory functions than CD56dim CD16+ NK cells. However, recent studies have reported that cytolytic activity of CD56bright NK cells increases when these cells are stimulated with IL-15or the combination of IL-12, IL-15 and IL-18. Previous studies from our center have shown that daily administration of low-dose IL-2 in patients with chronic graft-versus-host disease (cGVHD) induces selective expansion of CD4+FoxP3+Helios+ regulatory T cells and CD56bright NK cells and improves clinical manifestations of cGVHD. The function of CD56bright NK cells expanded by low-dose IL-2 has not previously been studied. Methods: Single cell mass cytometry (CyTOF) with a panel of 35 metal tagged antibodies was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from 10 adult patients with active cGVHD receiving daily low-dose IL-2 therapy. Patients in this clinical trial received extracorporeal photopheresis (ECP) for 8 weeks prior to starting daily low dose IL-2. ECP therapy (twice weekly) was continued when patients began low-dose IL-2 (1x106 IU/M2/day x 8 weeks). The analytic panel included 26 cell surface markers to identify distinct lymphocyte subsets and 9 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. NK cytolytic activity was measured in flow cytometry-based cytotoxicity assays. CD56bright and CD56dim NK cells from 8 adult patients were purified from cryopreserved PBMC by cell sorting and incubated with labeled K562 cells for 4 hours followed by staining with 7-AAD and Annexin-V. E:T ratio of 1:1 was used for incubation with K562 targets. Results: No changes in extracellular or intracellular NK cell markers or quantitative changes in NK cells were observed during the initial 8 week ECP treatment period. Selective expansion of CD56bright NK cells was noted after 1 week of IL-2 therapy (9W) and continued during 8 weeks of daily IL-2 therapy. Increased expression of NKp30, Nkp46, NKG2D, HLA-DR and Ki67 occurred in expanded CD56bright NK cells with peak expression at 1 week after starting IL-2 (9W). At later time points during IL-2 therapy, expression of NKG2D, HLA-DR and Ki67 returned to baseline (Figure 1A). Expression of CD56, CD122 and NKG2A continued to increase during IL-2 treatment. In contrast, expression of CD25 by expanded CD56bright NK cells decreased during IL-2 treatment. Expression of phosphorylated signaling proteins did not change in any NK cell subset during IL-2 treatment.　Cytolytic activity was measured in CD56bright and CD56dim NK cell subsets at different times during ECP and IL-2 therapy. After thawing, flow-sorted NK cell subsets were cultured for 16-20 hours with IL-2 (100 IU/ml). Cells were then washed and incubated with tumor targets (K562) for 4 hours and % killing was assessed by flow cytometry. Compared to pre IL-2 treatment, cytolytic activity of CD56bright NK cells increased during IL-2 treatment while cytotoxicity of CD56dim NK cells did not change. Notably, cytotoxicity of CD56bright NK cells became significantly higher than CD56dim NK cells during IL-2 therapy (Figure 1B). Conclusion: Single cell mass cytometry revealed that daily low dose IL-2 therapy induces selective expansion, activation and increased expression of activating NK receptors in CD56bright NK cells. CD56dim NK cells were not affected by IL-2 therapy. In vitro assays revealed that cytolytic activity of CD56bright NK cells increased during IL-2 treatment and exceeded the cytotoxicity of CD56dim NK cells. CD56bright NK cells, traditionally considered to be minimally tumor-responsive, are effectively stimulated by daily low dose IL-2 exposure to enable potent cytotoxicity in response to tumor targets. In patients receiving low-dose IL-2 after allogeneic HSCT, expanded CD56bright NK cells may contribute to graft versus leukemia (GVL) and help prevent relapse after transplant. Disclosures Nikiforow: Kite Pharma: Consultancy. Ho:Jazz Pharmaceuticals: Consultancy. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Simian Immunodeficiency Virus Infection Induces Expansion of α4β7+ and Cytotoxic CD56+ NK Cells

2010 ◽  
Vol 84 (17) ◽  
pp. 8959-8963 ◽  
Author(s):  
R. Keith Reeves ◽  
Tristan I. Evans ◽  
Jacqueline Gillis ◽  
R. Paul Johnson
Keyword(s):  
Nk Cells ◽  
Simian Immunodeficiency Virus ◽  
Cell Activation ◽  
Cell Function ◽  
Nk Cell ◽  
Dual Function ◽  
Cd107a Expression ◽  
Immunodeficiency Virus ◽  
Siv Infection ◽  
Ifn Γ

ABSTRACT Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker α4β7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, α4β7 expression was associated with increased NK cell activation and, on CD16+ NK cells, delineated a unique dual-function cytotoxic-CD107a+/gamma interferon (IFN-γ)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56+ NK cells, α4β7+ and α4β7− NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.

scholarly journals The IRE1-XBP1s Pathway Impairment Underpins NK Cell Dysfunction in Hodgkin Lymphoma, That Is Partly Restored By PD-1 Blockade

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2795-2795
Author(s):  
Karolina Bednarska ◽  
Jay Gunawardana ◽  
Frank Vari ◽  
Qingyan Cui ◽  
Gayathri Thillaiyampalam ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Function ◽  
Nk Cell ◽  
Advisory Committees ◽  
Pathway Activation ◽  
Pathway Inhibition ◽  
The Impact

Background The frequent loss of Major Histocompatibility Complex I molecule (MHC-I) on Hodgkin/Reed-Sternberg (HRS)-cells renders them susceptible to Natural Killer (NK) cell-mediated lysis in Hodgkin Lymphoma (HL). Optimal NK cell function involves migration from peripheral blood to sites of disease, formation of an immune synapse (NKIS) between NK cells and HRS cells, and release of effector molecules. We recently showed that PD-1+ NK cells are expanded in the circulation of patients with HL (Vari, F Blood 2018), and that PD-1 blockade enhances their anti-HRS capabilities. However, mechanisms behind the functional impairment of NK cells in HL patients and the impact PD-1 blockade has on NK cell function remain to be established. Although the IRE1-XBP1s pathway, part of the unfolded protein response (UPR) system, has established and fundamental roles in macrophage, B, T and dendritic cells homeostatic function, its involvement in NK cells remains unknown. We hypothesized that IRE1-XBP1s dysfunction contributes to NK cell impairment and tested the impact of PD-1 blockade on individual components of NK cell function, including migration, NKIS formation, and cytokine release. Methods Ex-vivo functional assays were performed on blood from 20 participants. Confocal microscopy, time-lapse imaging, trans-well migration, and functional in-vitro immune assays were utilized on a range of NK and HRS cell lines, with and without IRE1-XBP1s small molecule inhibitors (4µ8c and 6-bromo) and/or PD-1 blockade (pembrolizumab). Results Stimulation of both NK cell lines and primary NK cells, with HRS lines resulted in marked and rapid IRE1-XBP1s pathway activation. This occurred independently of the canonical UPR and was associated with increased NK cell effector function. However, IRE1-XBP1s pathway inhibition resulted in aberrant NK cell morphology, reduced motility and migration, deficient NKIS formation and impaired interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) release. Next, we tested the IRE1-XBP1s pathway in the pre-therapy blood of patients with HL and compared this with healthy age/gender matched controls. Strikingly, following co-culture with an HRS-line the pathway was not activated, but this abnormality was restricted to the CD56brightCD16-ve subset that we have previously shown to be expanded and enriched in PD-1 (as well as downregulation of the lymphoid migratory chemokine CCR7) in patients with HL. In subsequent experiments using in-vitro expanded populations of primary NK cells from HL patients, IRE1-XBP1s pathway inhibition impaired the migration, NKIS formation, CD107a degranulation, and secretion of IFNγ and TNFα. Effects were partially but not completely restored by addition of PD-1 blockade (Fig 1). Conclusion Here, we outline a hitherto unrecognized mechanism involving the IRE1-XBP1s pathway that is pivotal to NK cell function, including the relatively poorly understood processes of migration, NKIS formation, and cytokine secretion. Notably, IRE1-XBP1s pathway activation is dysfunctional within the PD-1 enriched CD56brightCD16- NK cell subset. Although PD-1 blockade appears to have a multi-faceted beneficial role on NK cell migrational/NKIS and cytokine release capabilities, it is still only capable of partial restoration of NK cell effector function. Further understanding of the pathways operative in NK cells may result in improved immunotherapeutic strategies to enhance this arm of the immune response in patients with HL. Disclosures Gandhi: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals First Evidence of Restoration of T and NK Cell Compartment after Venetoclax Treatment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1860-1860 ◽  
Author(s):  
Iris de Weerdt ◽  
Tom Hofland ◽  
Johan Dobber ◽  
Julie Dubois ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cytokine Production ◽  
Nk Cell ◽  
Advisory Committees ◽  
Ifn Γ

Abstract Introduction Chronic lymphocytic leukemia (CLL) is characterized by a profound immune suppression. In addition, CLL cells evade immune destruction by interacting with cells of the adaptive immune system, resulting in dysfunctional T cells. CD4+ T cells are skewed towards a TH2-profile and the number of regulatory T (Treg) cells, that diminish cellular immune responses, is increased in CLL patients. CD8+ T cells resemble exhausted T cells and have reduced cytotoxic, yet increased cytokine production capacity. The cytotoxic function of NK cells is impaired in CLL patients, but in contrast to CD8+ T cells their cytokine production is also compromised, presumably induced by CLL cells. These data are chiefly obtained from studies on peripheral blood (PB). Although the lymph node (LN) compartment has a central role in the pathobiology of CLL, very little is known about the composition of non-malignant lymphocytes in LN tissue. The Bcl-2 inhibitor venetoclax (Ven) is highly effective in CLL and, especially in combination with anti-CD20 monoclonal antibodies such as obinutuzumab (O), results in high rates of minimal residual disease (MRD) undetectable responses. However, the prospective effects of venetoclax on non-malignant lymphocytes in patient samples remain largely unexplored. Methods PB and LN biopsy specimens were collected at baseline from patients enrolled in the 1st-line FCR-unfit HOVON 139 / GIVE trial. Study treatment consisted of O (cycle 1-2), Ven+O (cycle 3-8) and Ven (cycle 9-14). Immune composition was analyzed by 7-color flow cytometry. Baseline PB samples were compared to paired LN samples. Moreover, PB samples of the first patients that completed 6 cycles of Ven monotherapy (cycle 14) were compared to baseline. Cytokine production and degranulation of T and NK cells was studied after stimulation of PBMCs with PMA/Ionomycin. Results Comparison of LN (n=28) vs PB (n=48) revealed a larger proportion of T cells in LN (13.2% vs 5.1% of the lymphocytes), at the expense of CLL cells, with a skewed CD4:CD8 ratio (5.2 in LN vs 1.8 in PB). Within the CD4+ T cells, significantly higher levels of both follicular T helper cells (15. 7% vs 5.2%) and Tregs (11.5% vs 6.9%) were found in LN (see Table). CD4+ T cells mostly consisted of naïve and memory T cells in both PB and LN. There were fewer CD8+ T cells and especially fewer effector CD8+ T cells in the LN in comparison to PB. CD8+ T cells in LN mostly had a naïve and memory phenotype. An increased percentage of LN-residing CD8+ T cells expressed the exhaustion marker PD-1 as compared to PB CD8+ T cells (30.4% in LN vs 12.4% in PB). We then compared PB baseline samples to PB obtained after cycle 14 (n=11). Ten patients achieved MRD undetectable levels (<10-4, determined by flow cytometry) and 1 patient was MRD intermediate (10-4-10-2). As expected, the treatment regimen led to complete elimination of CD19+ B cells. In contrast, absolute numbers of CD4+ and CD8+ T cells did not change during treatment. Differentiation status of CD4+ and CD8+ T cells remained similar. Interestingly, the proportion and absolute number of Tregs decreased after treatment (6.1% vs 0.9% of CD4+ T cells). After stimulation with PMA/Ionomycin, the percentage of IL-2 producing CD4+ T cells increased after treatment, leading to a higher IL-2:IL-4 ratio, that suggests normalization towards a TH1-profile. Fewer CD8+ T cells expressed PD-1 after treatment. The fraction of CD8+ T cells that produced IFN-γ (69.8% vs 56.2%) and TNF-α (58.4% vs 40.3%) decreased. Degranulation of CD8+ T cells did not change upon treatment. After treatment, the capacity of NK cells to degranulate increased. In addition, a larger proportion of NK cells produced IFN-γ, suggesting recovery of NK cell function after treatment. Conclusion In conclusion, our data strengthen the view that CLL cells reside in an immune suppressive environment in the LN. Moreover, we provide the first evidence that the Ven+O regimen does not harm non-malignant lymphocyte populations other than B cells. Both the improved cytokine production of NK cells and diminished cytokine production of CD8+ T cells may point to normalization of immune function. Collectively, the phenotypical and functional changes observed may reflect the eradication of the immunosuppressive CLL clone by Ven+O and subsequent recovery of the immune microenvironment in CLL patients. Disclosures Eldering: Celgene: Research Funding. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Levin:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kater:Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals GTB-3550 TriKE™ for the Treatment of High-Risk Myelodysplastic Syndromes (MDS) and Refractory/Relapsed Acute Myeloid Leukemia (AML) Safely Drives Natural Killer (NK) Cell Proliferation At Initial Dose Cohorts

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-8 ◽  
Author(s):  
Erica D. Warlick ◽  
Daniel J. Weisdorf ◽  
Daniel A. Vallera ◽  
Rose Wangen ◽  
Dixie Lewis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nk Cells ◽  
Continuous Infusion ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Activation ◽  
Nk Cell ◽  
Absolute Lymphocyte Count ◽  
Advisory Committees ◽  
Dose Levels

Introduction: Relapsed/refractory AML and MDS present a clinical challenge. Despite FDA approval of multiple new targeted agents, many patients lack actionable mutations and have exhausted conventional chemotherapeutic options. We have shown that NK cell infusions after lymphodepleting chemotherapy can induce remissions in relapsed/refractory AML. However, this cell-based therapy lacks antigen specificity. To address this, we developed GTB-3550 TriKE, a novel CD16/IL-15/CD33 Tri-Specific Killer Engager (TriKE). GTB-3550 TriKE is comprised of two single chain variable fragments (scFvs); one that binds CD16 on NK cells and one that binds CD33 on myeloid malignancies, plus an IL-15 linker bridging the CD16 and CD33 scFvs for sustained cell activation. Pre-clinical data shows specific NK cell activation and targeted cytolytic activity in xenogeneic AML mouse models. Methods: Patients with CD33+ malignancies (primary induction failure or relapsed AML with failure of one reinduction attempt or high risk MDS progressed on two lines of therapy) age 18 and older were eligible (NCT03214666) if they had adequate renal, hepatic, cardiac and lung function and absolute lymphocyte count (ALC) ≥ 200 cells/µL or absolute circulating CD56+/CD3- NK cell count &gt;25 cells/µL within the 14 days prior to start of therapy. The primary endpoint is to identify the maximum tolerated dose (MTD) of GTB-3550 TriKE. Correlative objectives include the number, phenotype, activation status and function of NK cells and T cells. During phase 1, each patient received the GTB-3550 TriKE at 5-200 mcg/kg/day (in 6 cohorts) for 3 weeks (infusion block #1, #2, and #3) of 96 hours continuous infusion separated by a 72-hour rest. Two patients enrolled per dose cohort. Disease response assessed by bone marrow biopsy between Days 21-42. Results: Four patients have enrolled, two at 5 mcg/kg/day and two at 10 mcg/kg/day, and three have completed therapy. Two patients with stable disease and one had substantial disease progression in the setting of a FLT-3 ITD mutation (Figure 1A). The first patient at 5 mcg/kg/day had stable disease after course 1 and qualified for retreatment with a second course of 3 weeks of GTB-3550 TriKE due to improved platelet transfusion requirements. Of the four complete courses of treatment delivered, all were delivered on schedule without treatment interruptions. The previous MTD of continuous infusion rhIL-15 was 2 mcg/kg/day (Conlon et al, Clin. Ca Res.) with associated fevers, tachycardia and constitutional symptoms. Validating our pre-clinical data showing decreased IL-15 potency when sandwiched between the CD16 and CD33 scFvs, patients treated with GTB-3550 TriKE treatment displayed no signs of clinical immune activation or SAE's in the 5 and 10 mcg/kg/day cohorts. Correlative studies have shown reproducible NK cell activity in all patients. NK cell activation (CD69+) increases early during treatment (Figure 1B). The greatest NK cell proliferation (Ki-67+) starts at day 3, is maximal at Day 8, and maintained above baseline at Day 15 and Day 22 (Figure 1C). This correlated with an increase proportion and absolute number of NK cells during treatment (Figure 1D). Targeted delivery of IL-15 to NK cells through the anti-CD16 engager showed preferential proliferation of NK cells and significantly less effect on CD8+ T cells. Additionally, the frequency of NK cells and absolute lymphocyte count decreased early during the continuous infusion of the GTB-3550 TriKE, which rebounded after the 72-hour rest period to higher levels than the pre-treatment baseline. Conclusion: This novel GTB-3550 TriKE administered by continuous infusion led to NK cell proliferation in all patients at initial dose levels and no clinically significant targeted toxicity. Though no objective responses were seen at the initially low dose cohorts tested, these early data suggest proof of principle that the drug has immune activity in humans. Subsequent patients given higher dose levels will determine if NK cell proliferation translates into clinical efficacy. Disclosures Weisdorf: Incyte: Research Funding; FATE Therapeutics: Consultancy. Vallera:GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Schroeder:GT Biopharma: Consultancy, Current equity holder in publicly-traded company. Felices:GT Biopharma: Consultancy. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: GTB-3550 TriKE is a cytokine immune engager binding to CD16 on NK cells, CD33 on myeloid blasts and IL-15 between the two engager components.

scholarly journals Cytokine-Mediated Activation of NK Cells during Viral Infection

2015 ◽  
Vol 89 (15) ◽  
pp. 7922-7931 ◽  
Author(s):  
Bailey E. Freeman ◽  
Hans-Peter Raué ◽  
Ann B. Hill ◽  
Mark K. Slifka
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Activation ◽  
Cell Function ◽  
Viral Infections ◽  
Nk Cell ◽  
Mcmv Infection ◽  
Cell Responses ◽  
Ifn Γ ◽  
Cytokine Stimulation

ABSTRACTNatural killer (NK) cells provide a first line of defense against infection via the production of antiviral cytokines and direct lysis of target cells. Cytokines such as interleukin 12 (IL-12) and IL-18 are critical regulators of NK cell activation, but much remains to be learned about how cytokines interact to regulate NK cell function. Here, we have examined cytokine-mediated activation of NK cells during infection with two natural mouse pathogens, lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV). Using a systematic screen of 1,849 cytokine pairs, we identified the most potent combinations capable of eliciting gamma interferon (IFN-γ) production in NK cells. We observed that NK cell responses to cytokine stimulation were reduced 8 days after acute LCMV infection but recovered to preinfection levels by 60 days postinfection. In contrast, during MCMV infection, NK cell responses to cytokines remained robust at all time points examined. Ly49H-positive (Ly49H+) NK cells recognizing viral ligand m157 showed preferential proliferation during early MCMV infection. A population of these cells was still detected beyond 60 days postinfection, but these divided cells did not demonstrate enhanced IFN-γ production in response to innate cytokine stimulation. Instead, the maturation state of the NK cells (as determined by CD11b or CD27 surface phenotype) was predictive of responsiveness to cytokines, regardless of Ly49H expression. These results help define cytokine interactions that regulate NK cell activation and highlight variations in NK cell function during two unrelated viral infections.IMPORTANCENatural killer cells play an important role in immunity to many viral infections. From an initial screen of 1,849 cytokine pairs, we identified the most stimulatory cytokine combinations capable of inducing IFN-γ production by NK cells. Ly49H+NK cells, which can be directly activated by MCMV protein m157, preferentially proliferated during MCMV infection but did not show enhanced IFN-γ production following directex vivocytokine stimulation. Instead, mature CD11b+and/or CD27+NK cells responded similarly to innate cytokine stimulation regardless of Ly49H expression. Collectively, our data provide a better foundation for understanding cytokine-mediated NK cell activation during viral infection.

Bi-Specific Killer Cell Engagers (BiKEs) Signaling Through CD16 and Targeting CD33 (CD16 × CD33), Triggers Natural Killer (NK) Cell Cytotoxic and Cytokine Production Against Refractory Acute Myeloid Leukemia (AML)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 256-256
Author(s):  
Andres Wiernik ◽  
Bree Foley ◽  
Bin Zhang ◽  
Michael R. Verneris ◽  
Erica Warlick ◽  
...  
Keyword(s):  
Nk Cells ◽  
Target Cell ◽  
Cell Activation ◽  
Cytokine Production ◽  
Nk Cell ◽  
Negative Control ◽  
Advisory Committees ◽  
Tnf Α ◽  
Ifn Γ ◽  
Survival Assay

Abstract Abstract 256 AML accounts for a large number of annual deaths due to leukemia worldwide. NK cells are effectors of the innate immune system that mediate the graft versus leukemia (GVL) effect in patients with AML and other hematologic malignancies. The safety and success of using haploidentical NK cell infusions to treat patients with AML has been previously demonstrated, but this therapeutic approach has limitations of potency and lacks specificity for leukemic targets. NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) usually occurs trough the binding of the activating receptor FcγRIIIA (also known as CD16) to the Fc portion of antibodies. CD16 is expressed on most CD56dim NK cells and induces NK cell activation, leading to interferon (IFN-γ) and tumor necrosis factor (TNF-α) secretion. CD16 shedding occurs upon NK cell activation, an effect that we have shown is mediated by a specific metalloproteinase called a disintegrin and metalloproteinase-17 (ADAM-17). We hypothesized that a BiKE antibody molecule, developed specifically to signal through CD16 and targeting the myeloid differentiation antigen CD33 (i.e., CD16 × CD33 BiKE) would enhance NK cell function against AML. In addition, we predicted that selective inhibition of ADAM-17 activity would prevent CD16 shedding and enhance the activity of the CD16 × CD33 BiKE. Bone marrow (BM) samples from 10 patients with primary refractory AML were obtained from our leukemia tissue bank and used as targets. CD33 was expressed on 8 of the 10 BM samples. Purified NK cells from healthy donors were isolated and incubated overnight in the absence of cytokines. NK cells and AML BM samples were treated with 1 ug/mL of bscFv CD16 × CD33 BiKE, scFv anti-CD16 (negative control) or DTCD33 × CD33 (anti-CD33 plus anti-CD33 spliced to truncated diphtheria toxin - negative control). NK cells under the above conditions were all treated with or without 5 uM of an ADAM17 inhibitor (Incyte). After 4 hours in culture, intracellular CD107a degranulation assay and intracellular TNF-α and IFN-γ assays were performed. A MTS survival assay was also performed on the target cells. In 8 of 10 AML samples, NK cells significantly degranulated and secreted cytokines (TNF-α and IFN-γ) only when treated with the CD16 × CD33 BiKE reagent. The MTS survival assay confirmed significant AML target cell death in the presence of the CD16 × CD33 BiKE. Combined treatment with the CD16 × CD33 BiKE and the ADAM17 inhibitor led to a significant increase in cytokine TNF-α and IFN-γ secretion by NK cells when compared to treatment with CD16 × CD33 BiKE alone. Phenotypic analysis of the NK cells after treatment with the ADAM17 inhibitor revealed a significant decrease in CD16 shedding as predicted. Of note, no NK cell activity was triggered by the 2 AML BM samples that lacked CD33 expression arguing in favor of the specificity of this molecule for CD33 positive AML. We then analyzed the ability of NK cells to kill multiple targets in the presence of the CD16 × CD33 BiKE over time. NK cells and HL60 cells (CD33 positive targets) were treated with 1 ug/mL of bscFv CD16 × CD33 BiKE and incubated overnight in the presence or absence of the ADAM17 inhibitor. On the following day, a second target exposure with chromium (Cr-) labeled HL60 cells was added to the culture in order to visualize the effect of NK cells on second targets. After 4 hours, intracellular CD107a, TNF-α, IFN-γ and standard Cr- release assays were performed. In the presence of the CD16 × CD33 BiKE, NK cells showed significantly more degranulation killing and secreted more cytokines in response to secondary targets. Cytokine secretion was also enhanced by the addition of the ADAM17 inhibitor to the BiKE. Collectively, these findings support the ability of a CD16 × CD33 BiKE to trigger NK cell activation through direct signaling of CD16, inducing secretion of cytokines, lytic granules and causing target cell death in resistant AML BM samples and HL60 targets. BiKE enhanced AML killing occurs over a wide range of CD33 target cell density as long as some expression is present. In addition, targeting ADAM17 prevents activation induced CD16 shedding and enhances NK cell cytokine production when combine with therapeutic antibodies. NK cell directed therapy by these compounds could specifically enhance the anti-leukemic potency and efficacy of NK cell adoptive therapy against myeloid disorders. Disclosures: Miller: Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

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